Single cell microorganisms (“microbes”) are routinely identified by mass spectrometric procedures in hundreds of microbiological labs all over the world, for instance for clinical diagnostics of infections, hygiene control in hospitals or bathing-establishments, or food analytics. Microbes may comprise bacteria, yeasts (single cell fungi), algae, or protozoa (e.g., plasmodia as pathogens of malaria). For all these microbes, identification procedures can be applied which utilize mass spectra of the essential cell components and compare those with reference spectra. In practice, this procedure is similar for all single cell species.
Hyphal fungi always form a mycelium. A mycelium is a mixture of fine threads, called “hyphae”, forming chains of cells with one or more nuclei. The threads may form branches, and may be woven in a complex manner. Any growth takes place only at the tips of the hyphae, in contrast to the growth of algae threads which grow by cell division inside the threads. The terms “hyphal fungi” or “mycelium forming fungi” will here be used to separate these multi-cell fungi from single cell fungi like yeast.
Up to now, the mass spectrometric identification procedures fail to identify hyphal fungi, because the cells of the mycelium show fast differentiation, mainly concerning the metabolism which will be quickly adapted to environmental conditions. Cells differentiated in different ways show, after cell lysis, mass spectra which usually appear to be surprisingly different. Spores, fruit bodies, colonies in soil, or from surfaces of wood, cheese, bread or wall paper show mass spectra which, for the same fungus, are quite different. In most cases, they show relatively few outstanding mass signals. It has become known that relatively safe identifications may be achieved by limiting the analyses to spores only as a selected differentiation variety. It is, however, disadvantageous to have to wait during cultivation for the formation of spores which may last weeks or even many months. Besides, there are sometimes several forms of spores for the same fungus like asci spores, basidia spores, conidias, and others with different mass spectra in most cases.
Any identification is, in principle, the determination of the species and thus the positioning in the taxonomical hierarchy, reaching from the uppermost rank, the domain (archaea, bacteria, eukariota) down to order, family, genus, and species. Fungi form their own kingdom besides plants and animals. The result of any successful identification is the name of the species; this name offers access to all written information about this species.
Identification of hyphal fungi is essential in many areas: building trade, maintenance of woods or farm land, clinical diagnostics, food production, material storage, home hygiene and many others. The identification of fungi may become as essential as the identification of bacteria. Most interestingly, there are more than a hundred thousand known and described species of fungi, in contrast to only some ten thousand described species of bacteria.
In the last years, for microbes like bacteria, archaea, yeast, or single-cell algae, there have become known several bio-molecular procedures of identification, like DNA or RNA sequencing or mass spectrometric measurement of cell ingredients. These procedures proved to be much faster and safer than classical identification procedures, with better rates for specificity (correct negative identification rate), sensitivity (correct positive identification rate) and other statistical error rates. It is highly desirable to develop similar procedures for fungi. Up to now, DNA sequencing has not really applicable because there are no unique sequence sections found for unique identifications, and often the polymerase chain reaction fails in the presence of fungus cell components; in contrast to the early international commitment to the 16S-sequences of ribosomal RNA (rRNA) for the unique identification of bacteria. Mass spectrometry is strongly affected by the strong and fast differentiation by adaption of the metabolism to environmental conditions.
The identification of bacteria by mass spectrometry is presented in some detail in the review article of van Baar (FEMS Microbiology Reviews, 24, 2000, 193-219: entitled “Characterization of Bacteria by Matrix-assisted Laser Desorption/Ionization and Electrospray Mass Spectrometry”). The identification is performed by similarity analyses between a spectrum of the bacterium to be identified with well-known reference spectra. For each similarity comparison with a reference spectrum, a similarity value is calculated. A bacterium may be regarded as identified if the similarity value for a distinct reference spectrum shows a clearly better similarity than the similarity values for all other reference spectra, and, in addition, a better value than a preselected similarity threshold.
The generation of mass spectra of microbes usually starts with the cultivation of clearly separated colonies (an “isolate”) on a gelatinous culture medium in a Petri-dish. With a small swab, e.g., a wooden toothpick, a small amount of bacteria from the colony is spotted onto a mass spectrometric sample plate. The cells are lysed in a well-known way, a solution of matrix material is added and dried, and the sample plate is inserted into the ion source of a time-of-flight mass spectrometer (TOF) operated with ionization by matrix-assisted laser desorption (MALDI). Ions are generated by pulsed laser shots, and their flight time is measured. Usually hundreds of single spectra are added together to improve the signal-to noise ratio. The term “mass spectrum of a microbe” or in short “sample spectrum” always refers to this sum spectrum, added together from many single mass spectra.
As already mentioned, the identification is based on similarity analyses of sample spectra with reference spectra from a library. There are different kinds of similarity analysis procedures, dependent on the amount and quality of data stored in the reference spectra. A spectrum may include pairs of masses and intensities of ions only, or may contain additional information like widths and variations of widths of the mass signals, variations of the intensity values, percentage of appearance of a signal above detection limit, and so on. The literature shows a variety of different similarity calculation procedures, some aiming for fast calculations, others aiming for high identification quality. The assignee of the present invention, Bruker Daltronics, provides a fast and precise similarity analysis procedure (i.e., Bruker MALDI Biotyper™ identifier system) showing a high rate of success, as many independent studies were able to prove. This similarity procedure is essentially based on matching mass values, and less essentially on matching intensities.
The similarity values may be reduced, by a corresponding scale transformation, to easily recognizable numbers, for instance, to a maximum similarity (identity of spectra) with a similarity value of 3.00. The transformation may even be performed in such a way, that a similarity value of 2.00 is the minimum value for a safe identification of the species. It is our experience, that such a scale has a high psychological value for the acceptance of the procedure.
Hyphal fungi can be cultured on agar in Petri-dishes in the same way as bacteria, with special kinds of agar with some antibiotics to prevent simultaneous growth of bacteria. Usually a sample is swabbed onto the agar, sometimes resulting in a chaotic growth of isolates superimposing each other. Some tiny amounts of mycelium of these colonies may then be transferred to new agar plates. After a relatively short time, the growing colony already shows some differentiation of the mycelium: mycelium from the edges of the colony show mass spectra which are different from mycelium from the center. Thus the colonies on agar are not the best basis for mass spectrometric identification. In addition, picking pure mycelium for the acquisition of mass spectra sometimes is difficult; some agar picked with the mycelium may disturb sample preparation and spectrum acquisition.
There is a need for a bio-molecular, preferably a mass spectrometric procedure for the fast and safe identification of hyphal fungi, with an unequivocal determination of the species within one or two days.